The Effect of Metformin in Diabetic and Non-Diabetic Rats with Experimentally-Induced Chronic Kidney Disease

Abstract

This work aimed to investigate whether treatment with the antidiabetic drug metformin would affect adenine-induced chronic kidney disease (CKD) in non-diabetic rats and rats with streptozotocin (STZ)-induced diabetes. Rats were randomly divided into eight groups, and given either normal feed, or feed mixed with adenine (0.25% w/w, for five weeks) to induce CKD. Some of these groups were also simultaneously treated orally with metformin (200 mg/kg/day). Rats given adenine showed the typical signs of CKD that included detrimental changes in several physiological and traditional and novel biochemical biomarkers in plasma urine and kidney homogenates such as albumin/creatinine ratio, N-acetyl-beta-D-glucosaminidase, neutrophil gelatinase-associated lipocalin, 8-isoprostane, adiponectin, cystatin C, as well as plasma urea, creatinine, uric acid, indoxyl sulfate, calcium, and phosphorus. Several indices of inflammation and oxidative stress, and renal nuclear factor-κB and nuclear factor erythroid 2-related factor 2 levels were also measured. Histopathologically, adenine caused renal tubular necrosis and fibrosis. The activation of the intracellular mitogen-activated protein kinase signaling pathway was inhibited in the groups that received metformin and STZ together, with or without adenine induced-CKD. Induction of diabetes worsened most of the actions induced by adenine. Metformin significantly ameliorated the renal actions induced by adenine and STZ when these were given singly, and more so when given together. The results suggest that metformin can be a useful drug in attenuating the progression of CKD in both diabetic and non-diabetic rats.

Keywords: adenine; chronic kidney disease; diabetes; metformin; rats.

Figure 1

The fasting blood glucose level in control rats, and rats treated with metformin (MF), adenine (A) and streptozotocin (STZ) alone or in combination. Each column and vertical bar represent mean ± SEM (n = 6). Differences between the groups were assessed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test, where p < 0.05. ^a denotes significant difference between control group vs. different groups; ^b denotes significant difference between the group given A alone vs. other groups treated with A; ^c denotes significant difference between the group given STZ alone vs. other groups treated with STZ; ^d denotes significant difference between the group given A + STZ vs. other groups treated with A + STZ; ^e denotes significant difference between the group given MF only vs. other groups treated with MF.

Figure 2

The renal concentration of nuclear factor-kappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) in control rats, and rats treated with metformin (MF), adenine (A), and streptozotocin (STZ) alone or in combination. Each column and vertical bar represent mean ± SEM (n = 6). Differences between the groups were assessed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test, ^a denotes significant difference between control group vs. different groups; ^b denotes significant difference between the group given A alone vs. other groups treated with A; ^c denotes significant difference between the group given streptozotocin (STZ) alone vs. other groups treated with STZ; ^d denotes significant difference between the group given A + STZ vs. other groups treated with A + STZ; ^e denotes significant difference between the group given MF only vs. other groups treated with MF.

Figure 3

Representative light microscopy images of H&E-stained kidneys sections (Bar = 100 µm). (A) Control group showing normal structures and architecture of renal tissue with intact glomeruli and renal tubules (Score 0); (B) rats treated with adenine (A) showing cystic dilatation of multiple renal tubules (asterisks), renal tubular necrosis with pyknotic nuclei (arrowheads), marked basophilia, and dilatation of bowman’s capsule (arrow) (Score 4); (C) rats treated with streptozotocin (STZ)-induced diabetes showing group cystic dilation of few renal tubules (asterisk) (Score 2); (D) rats treated with adenine and STZ showing, renal tubular necrosis (arrowheads), cystic dilatation of renal tubules (asterisks), and cellular casts in renal tubules (arrow) (Score 4); (E) rats treated with metformin (MF) showing normal glomerulus and renal tubules (Score 0); (F) rats treated with adenine and MF showing renal tubular dilatations (asterisks) and tubular necrosis with marked basophilia (arrowhead) (Score 3); (G) rats treated with STZ and MF showing normal histological structures of the majority of the renal tubules, intact glomeruli and few dilated tubules (asterisk) (Score 1); (H) rats treated with adenine, STZ and MF showing normal renal tubules and intact glomeruli with few dilated tubules (Score 0).

Figure 4

Representative photographs of Picro-Sirius red staining on sections of renal tissues (Bar = 100 µm) from control rats and rats treated with adenine (A), streptozotocin (STZ), and metformin (MF), alone or in combination. (A) Control and (E) MF groups showed normal kidney architecture and histology. (B) adenine (A,C) STZ showed stained fibrotic areas fibers (arrowheads) and yellow non-collagen structures. (D) A + STZ groups showed changes in normal kidney architecture and histology. (F) The A + MF group showed fibrotic areas fibers (arrowheads). (G) STZ + MF and (H) A + STZ + MF groups showed an improvement in the histologic appearance and a significant decrease in fibrotic areas fibers when compared with the STZ and A + STZ group, respectively.

Figure 5

Representative photographs of Periodic Acid–Schiff (PAS) staining on sections of renal tissues (Bar = 100 µm) from control rats and rats treated with adenine (A), streptozotocin (STZ), and metformin (MF), alone or in combination. Control rats and rats treated with MF only ((A,E), respectively) showed normal renal histology. (B) Adenine (A,D) A + STZ alone treated groups shows tubular atrophy represented by a thickened tubular basement membrane (arrow) stained positive (magenta red). (C) STZ (STZ) group shows only a slight change in tubular atrophy, when compared to control group. (F) Adenine-MF (A + MF) group showing a thickened tubular basement membrane (arrow). (G) STZ + MF and (H) A + STZ + MF groups showed improvement in the histologic appearance and a significant decrease in tubular atrophy.

Figure 6

The renal concentration of phospho-p44 or phospho-p42 mitogen-activated protein kinase (MAPK) in control (CON) rats, and those treated with adenine (A, 0.25% w/w in feed), streptozotocin (STZ, 55 mg/kg by intraperitoneal injection), metformin (MF, 200 mg/kg/day), or a combination of these treatments. Each vertical column with bar represents the mean ± SEM (from 6 rats in each group). Differences between the groups were assessed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test, where p < 0.05 was considered significant. ^a denotes significant difference between control group vs. different groups; ^b denotes significant difference between the group given A alone vs. other groups treated with A; ^c denotes significant difference between the group given STZ alone vs. other groups treated with STZ; ^d denotes significant difference between the group given A + STZ vs. other groups treated with A + STZ; ^e denotes significant difference between the group given MF only vs. other groups treated with MF.